Regulation of EMT During Avian Heart Valve Formation

描述(申请人提供)：在心脏发育过程中，瓣膜前体细胞首先出现在房室管，这是转化生长因子介导的上皮-间充质细胞转化(EMT)的结果。以往的研究表明，这种EMT可以分为两个阶段，即激活阶段和侵袭阶段。阶段1部分由转化生长因子β2(TGFβ2)介导，阶段2部分由转化生长因子β3介导。先前的研究表明，5种不同的受体，即转化生长因子受体II、受体III、Endoglin、ALK2和ALK5的抑制可以阻断EMT。我们的假设是，还有两个离散的信号复合体介导了转化生长因子β调节的EMT。为了继续分析转化生长因子β对正常心脏发育的调节作用，第一个目的是研究特定的转化生长因子β亚型和受体在介导EMT调节中的相关性。基因芯片实验将评估特定的转化生长因子β亚型的缺失对EMT前组织、EMT期间和EMT后间充质细胞中基因表达变化的影响。然后，在受转化生长因子调控的基因中选择标记，用来评估当每个特定受体被破坏时，基因表达是否发生变化。这些数据之间的关联将指向介导EMT的特定受体和配体信号复合体。这些假定的复合体将在蛋白质水平得到证实。来自其他实验室的数据表明，心脏EMT中存在多种信号转导机制和细胞内调节因子，包括ErbB2、NF-1、VEGF和NFATc1。在第二个目标中，将进行实验以确定是否干扰转化生长因子信号改变了任何其他候选机制，或者这些分子的丢失是否改变了转化生长因子配体或受体的表达。在神经脊系统中观察到，SOX8、9或10在神经管中的强迫表达会导致EMT，并产生异位的神经脊细胞。我们推测，这类似于心脏中的细胞侵袭步骤，需要一些转化生长因子β的活性。在第三个目标中，将进行实验，以确定外源性SOX基因的表达是否会迫使EMT，以及SOX基因的表达是由转化生长因子信号转导调节还是调节转化生长因子介导的反应。总之，这些实验将为EMT调节机制提供新的线索，并有助于理解先天性心脏病。

DESCRIPTION (provided by applicant): During cardiac development, valve progenitors first arise in the atrioventricular canal as a result of a TGF¿-mediated epithelial-mesenchymal cell transformation (EMT). Prior studies showed that this EMT can be divided into two stages, an activation stage and an invasion stage. Stage 1 is mediated, in part, by transforming growth factor beta 2 (TGF¿2) and stage 2 is mediated, in part, by TGF¿3. Previous work showed that EMT can be disrupted by the inhibition of 5 different TGF¿ receptors, TGF¿ receptor II, TGF¿ receptor III, Endoglin, ALK2 and ALK5. It is our hypothesis that there are two more discrete signaling complexes mediating TGF¿-regulated EMT. To continue the analysis of TGF¿-mediated regulation of normal heart development, the first aim will examine the correlation between specific TGF¿ isoforms and receptors in mediating EMT regulation. Microarray experiments will be performed to assess the effect loss of specific TGF¿ isoforms on altered gene expression in tissues prior to EMT, during EMT and in mesenchymal cells after EMT. Selected markers among the TGF¿-regulated genes will then be used to assess whether gene expression is altered as each of the specific receptors is disrupted. Correlations between these data will point to specific receptor and ligand signaling complexes that mediate EMT. These putative complexes will be confirmed at the protein level. Data from other labs has implicated a variety of signal transduction mechanisms and intracellular regulators in cardiac EMT including ErbB2, NF-1, VEGF and NFATc1. In the second aim, experiments will be undertaken to determine whether disruption of TGF¿ signaling alters any of the other candidate mechanisms or whether loss of these molecules alters TGF¿ ligand or receptor expression. It was observed in the neural crest system that forced expression of Sox 8, 9 or 10 in the neural tube would cause an EMT and produce ectopic neural crest cells. We conjecture that this is analogous to the cell invasion step in the heart and that it requires some TGF¿ activity. In the third aim, experiments will be undertaken to determine whether exogenous expression of Sox genes will force EMT and whether Sox gene expression is regulated by TGF¿ signal transduction or regulates TGF¿-mediated responses. Together these experiments will shed new light on the mechanisms of EMT regulation and aid in an understanding of congenital heart disease.